Species-Level Identification of the Blowfly Chrysomya Megacephala and Other Diptera in China By
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Genome Species -level identification of the blowfly Chrysomya megacephala and other Diptera in China by DNA barcoding Journal: Genome Manuscript ID gen-2015-0174.R2 Manuscript Type: Article Date Submitted by the Author: 12-Jul-2016 Complete List of Authors: Qiu, Deyi ; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center Cook, Charles ; European Molecular Biology Laboratory, European BioinformaticsDraft Institute (EMBL-EBI), Wellcome Genome Campus YUE, Qiaoyun; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center, Hu, Jia; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center Wei, Xiaoya ; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center Chen, Jian; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center Liu, Dexing; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center Wu, Keliang; Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center Keyword: blowfly, haplotype network, invasive species, Diptera, pest https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 37 Genome Species-level identification of the blowfly Chrysomya megacephala and other Diptera in China by DNA barcoding Deyi Qiu 1, Charles E. Cook 2, Qiaoyun Yue 1, *, Jia Hu1, Xiaoya Wei 1, Jian Chen 1, Dexing Liu 1, and Keliang Wu 1 1. Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center, 2, Zhongshan 6 road, Zhongshan 528403, Guangdong, China 2. European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK * Corresponding author: [email protected],Draft [email protected] Conceived and designed the experiments: QY, DQ. Performed the experiments: QY, DQ, JH, XW, JC, DL, KW. Analyzed the data: QY, DQ, CEC. Wrote the paper: QY, CEC. Competing Interests: The authors have declared that no competing interests exist. gen-2015-0174.R2 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 37 Abstract The blowfly Chrysomya megacephala , or oriental latrine fly, is the most common human-associated fly of the oriental and Australasian regions. C. megacephala is of particular interest for its use in forensic entomology and because it is a disease vector. The larvae are economically important as feed for livestock and in traditional Chinese medicine. Identification of adults is straightforward, but larvae and fragments of adults are difficult to identify. We collected C. megacephala , its allies Chrysomya pinguis and Protophormia terraenovae, as well as flies from 11 other species from 52 locations around China, then sequenced 658 base pairs of the COI barcode region from 645 flies of all 14 species, including 208 C. megacephala , as the basis of a COI barcode library for flies in China. While C. megacephala and its closest relative C. pinguis are closely related (mean K2P divergence of 0.022), these species are completely non-overlapping in their barcode divergences, thus demonstrating the utility of the COI barcode region for the identification of C. megacephala . WeDraft combined the 208 C. megacephala sequences from China with 98 others from public databases and show that worldwide COI barcode diversity is low, with 70% of all individuals belonging to one of three haplotypes that differ by one or two substitutions from each other, reflecting recent anthropogenic dispersal from its native range in Eurasia. Keywords Haplotype network, blowfly, invasive species, Diptera, pest gen-2015-0174.R2 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 37 Genome Introduction The blowfly Chrysomya megacephala (Fabricius), or oriental latrine fly, is the most common human-associated fly of the oriental and Australasian regions (Wall and Shearer 1997). C. megacephala larvae develop in feces and decomposing flesh and consequently can be found at extremely high density (>95% of flies) under some environmental circumstances, such as locations near fish-processing activities (Wall et al. 2001). C. megacephala is native to Eurasia but through human action has spread around the world: by December 1975 it was reported from South America (Brazil) (Imbiriba et al. 1977) and later became established in New Zealand, Africa (Williams and Villet 2006), and then in North, South, and Central America via harbours and airports (Wells 1991; Williams and Villet 2006). It has a reported distribution across the whole of China except for arid high-elevation regions in Xinjiang, Qinghai, and Tibet (Xue and Zhao 1996). C. megacephala is of particular importance to humans for a range of reasons: 1) it is considered as one of the most importantDraft fly species in the science of forensic entomology (Cai et al. 2005; Goff 2001; Shi et al. 2008; Wu and Hu 2012; Xue and Zhao 1996); 2) in traditional Chinese medicine wuguchong, the dried larva of C. megacephala is believed to have the curative effect of clearing stagnant heat-toxicity from the human body (Luo 1993); 3) live larvae are used in medicine in the form of “maggot therapy” (Taha et al. 2010); 4) it is an important source of animal feed protein (Sing et al. 2012); 5) it can cause myiasis (or fly strike) in sheep and occasionally in humans as it can invade open wounds (Bunchu et al. 2007); and 6) C. megacephala is also a disease vector and is known to lay eggs on human feces and subsequently transmit diseases such as bacterial gastroenteritis if it comes into contact with human food (SukontasonDNA barcoding et al. 2007). has been successfully used for the molecular identification of a broad variety of insect taxa, including many Diptera (Nelson et al. 2007; Hernandez-Triana. 2015; Liao et al. Renaud et al. 2012; Rivera and Currie 2009; Schuehli et al. 2007), including C. megacephala and the closely related species C. pinguis (Nelson et al. 2012; Ramaraj et al. 2014; Salem et al. 2015)( . DNA barcoding, usually of a specific region in the mitochondrial cytochrome c oxidase subunit I (COI) gene, generally relies on the observation that intraspecific COI variation is usually lower interspecific variation (Raupach et al. 2014). Consequently, comparative sequence analyses typically, but not always, reveal a “barcoding gap” (Meyer and Paulay 2005) on plots of pairwise gen-2015-0174.R2 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 37 sequence differences and thereby allow molecular species-level identification of sequences generated from unidentified or unidentifiable samples, such as insect larvae or bloodstains (Hebert et al. 2003, 2004). DNA barcoding has been criticized as a single-character typological approach that cannot replace systematic science and will not work for all clades (DeSalle et al. 2005; Ebach 2011; Klausnitzer 2010; Will et al. 2005). Nevertheless, it has become an important, useful, and increasingly used tool for species descriptions (Butcher et al. 2012; Hendrich and Balke 2011; Stoev et al. 2010; Tamura et al. 2013; Wesener 2012; Wesener et al. 2011) as well as various other biological disciplines Adamowicz 2015), including forensics (Ferri et al. 2009; Meiklejohn et al. 2011), pest biology (Engstrand et al. 2010), Inspection and Quarantine (Liao et al. 2014; Liu et al. 2014; Wei et al. 2014; Yue et al. 2013), and conservation biology (Neigel et al. 2007; Ward et al. 2008). Examples are the recommendation of barcoding for identification of flightless weevils in the genus Trigonopterus as a substituteDraft for a traditional morphological key (Riedel et al. 2013); identifying the sources of food substitution or contamination (Cawthorn et al 2012 ); identifying the presence of genetically modified organisms (Barcaccia et al 2016 ); and identifying birds “minced” in jet engineers (Wong and Hanner 2008; Grant 2007). In sum, DNA barcoding has proven both useful and reliable for species identification, particularly for degraded or partial specimens, for many taxonomic groups. This identification is only possible, though, if data from reliably identified specimens are available in public databases. Adult C. megacephala are easily recognizable by experts, but less so for non-experts, while eggs, larvae, and fragments of adults, all of which may be encountered by pest control or public health workers, cannot be identified morphologically. A related question is whether C. exhibits any geographic structure that might allow assignment of place of origin to a sample of unknown provenance. Despite the ubiquity and economic importance of this species, C. megacephala barcode sequences, like those of many arthropods, are still poorly represented in public databases. In this study we collected C. megacephala as well as flies from 13 other species seven other genera, from 52 localities around China in order to confirm the utility of DNA for identification of the economically important C. megacephala and to establish a basic barcode library for C. megacephala and other related flies that co-occur with this species. There are 39 named species of the genus Chrysomya (http://eol.org/pages/56219/overview accessed 23 June gen-2015-0174.R2 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 37 Genome 2016), of which three are common in China: C. megacephala, C. pinguis, and C. phaonis . C. commonly co-occurs with C. megacephala and is the closest relative of C. megacephala (Yang, et 2014). We successfully collected individuals of C. pinguis and report the sequences here. We did identify any C. phaonis specimens from our sampling and therefore cannot yet report C. phaonis barcodes, but adults of C. phaonis are morphologically distinct from C. megacephala (Yang, et al 2014) and it is very unlikely that C. phaonis samples would be confused for C. megacephala . We will report C. phaonis barcode sequences if samples become available in future. We also compared C. megacephala COI barcode sequences from our work with other publicly available C. megacephala sequences to assess whether variation within China is comparable to worldwide variation in this species. Material and Methods Sample collection Draft Adult flies were collected with a sweep net from 52 different localities in China during the summers of 2012 and 2013. As this was not an ecological study, we did not undertake random transects.